ProjectSummary Chromatin,thepackagingforDNAintheeukaryoticnucleus,isadynamicentitythatisaffected bycellularprocessessuchasreplication,transcriptionandrepair.Ourviewsongeneregulation have evolved from simple notions about changes in DNA sequence, to a more complex perspective that takes into account multiple epigenetic factors such as chromatin structure, chromatincompositionandthree-dimensionalgenomeorganization.Overthelastdecade,much attention has been devoted to epigenetic alterations in chromatin, including post translational modifications of chromatin components, long non-coding RNAs that localize to and regulate genes, and most recently large scale changes in genome organization that affect interactions betweendistantgenomicregions.However,oneaspectofepigeneticgeneregulationthathas receivedasurprisinglysmallamountofattentionisthechangesinchromatinstructurethatoccur asaconsequenceoftranscription.Transcriptionispervasive,withover90%oftheeukaryotic genomeproducingRNAs.Thecommonchromatinstructuresthatresultfrom,anddependon transcription,suchasRloops,arelikelytocontributesignificantlytoepigeneticgeneregulation. But exactly how is not known. R loops are triplex nucleic acid structures formed during transcriptionwhenanRNA,eitheranmRNAoralongnon-codingRNA,invadesdsDNA,forming anRNA-DNAhybridandadisplacedssDNA.Undernormalconditions,Rloopsfunctioninall aspectsofgeneregulation,butaberrantformationofRloops,ormutationsincomponentsthat regulatethem,isassociatedwithseveralneurodegenerativediseasesandcancers.Rloopsare especially relevant in repeat expansion disorders where their unscheduled formation results in aberranttranscriptionanddiseasepathology.ButwhatcausesRloopstoformaberrantlyorbe resolved once created remains unknown. Identifying the molecular players that function at R loopsisanimportantsteptowardtargetingRloopsfortherapiesforneurologicaldisorders.We willdevelopnewmethodologiestoisolateandidentifyRloopregulators,createaframeworkof molecular tools to elucidate mechanisms of these regulators and finally undertake a molecular screening approach to identify factors that facilitate transcription through a pathogenic R loop formingrepeat.Asaresult,weexpecttoidentifynewregulatorsofRloopsthatcanleadtonovel therapeuticcandidatesforneurodegenerativerepeatexpansiondisorders.